Melanocytes are pigment producing cells located in the basal layer of the epidermis, the bulb of the hair sheaths, the choroid of the eye and the leptomeninges of the brain. As humans age, there is a progressive decline in melanocyte number and function in the skin, and hair and eye, while nothing is known about the melanocyte's fate in the brain. Loss of melanocytes in the ear and skin is associated with partial deafness and imbalance, and with skin cancers after the 5th decade of age respectively. Since the skin is constantly exposed to the environment, loss of epidermal melanocytes may be accentuated by environmental stress (ultraviolet radiation, heat, cold) which may result in early terminal differentiation/senescence of these cells. We were the first to develop an "in vitro" model for human adult epidermal melanocytes which allowed us to define a terminally differentiated melanocyte. We demonstrated that the potent cAMP inducer cholera toxin, induced a dramatic increase in melanin deposition in melanosomes, morphological changes and irreversible withdrawal from the cell cycle. We propose to extend these studies and characterize molecular pathways involved in melanocyte proliferation, terminal differentiation and senescence. We will analyze terminal differentiation by defining the molecular consequences of steady, high levels of cAMP and melanin content by the use of cAMP analogues and by the constitutive expression of activated Galphas proteins, kinetics of activation by phosphorylation of the cAMP responsive element binding protein CREB and the repressor CREM, and binding of CREB to the cAMP enhancer element. Using the yeast two-hybrid system, we will define novel, melanocyte specific CREB-protein interactions between CREB and CREB-repressors in proliferating and terminally differentiated melanocytes. We will determine also the contribution of the melanogenic enzymes tyrosinase, TRP-1, TRP-2 and Pmel 17 to the induction of terminal differentiation and their possible use as markers for terminal differentiation/senescence. In addition we will define similarities/differences between terminally differentiated and senescent melanocytes, by analyzing the expression and function of the cell cycle inhibitors p21/p27/p16/p15 and the proliferating cell nuclear antigen (PCNA) in both phenotypes, their modulation by ultraviolet radiation and by making stable melanocyte cell strains capable of inducible expression of sense and antisense sequences of these inhibitors. The studies proposed will be the basis for future studies aimed at understanding the role of melanocyte aging in the skin and also in other organs like the eye and the brain.